Anti-cancer therapeutic compositions containing whey protein concentrate

ABSTRACT

This invention relates to a method of treatment of patients having lesions resulting from cancer cells and to an application as an anti-cancer composition of undenatured whey protein concentrate.

RELATED APPLICATIONS

This application is a 371 of PCT/CA93/00309, filed Aug. 5, 1993 which isa Continuation-in-Part of U.S. Ser. No. 07/929,347, filed Aug. 13, 1992,now abandoned, which is a CIP of U.S. Ser. No. 07/417,246 filed Oct. 4,1989, now U.S. Pat. No. 5,290,571 which is a CIP of U.S. Ser. No.07/289,971, filed Dec. 23, 1988, now abandoned, which in turn, is a CIPof U.S. Ser. No. 07/188,276, filed Apr. 29, 1988, now abandoned. Thisapplication is also a CIP of U.S. application Ser. No. 07/866,756, filedApr. 10, 1992, now U.S. Pat. No. 5,456,924 which is a CIP of U.S.application Ser. No. 07/417,246 filed Oct. 4, 1989, now U.S. Pat. No.5,290,571 which is a CIP of said U.S. Ser. No. 07/289,971, filed Dec.23, 1988, now abandoned, which is a CIP of Ser. No. 07/188,276, filedApr. 29, 1988, now abandoned. The contents of those applications areincluded by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery thatundenatured whey protein concentrate has an enhanced immunologicaleffect. More specifically, this invention relates to the effect of theoral administration of undenatured whey protein concentrate (WPC) onhost resistance to the development of chemically induced cancer and alsothe effect of such oral administration on the inhibition of cancer.

In U.S. application Ser. No. 298,971 and Ser. No. 417,246, and also inBounous et al "Dietary Whey Protein Inhibits the Development ofDimethyl-hydrazine induced Malignancy" (1) we described experimentsshowing that continuous feeding of WPC in the diet inhibits thedevelopment (number and size of tumours) in the colon of a mouse over aperiod of 24 weeks of dimethylhydrazine (DMH) treatment. Thisanti-tumour effect could be caused by increased resistance of targetcells to the carcinogen and/or a direct inhibitory effect of WPC on thecancer cells. A subsequent series of experiments (2) where animals werefed Purina diet for the first 20 weeks of DMH and then switched to WPCdiet for the remaining 8 weeks of DMH treatment, suggested someinhibitory effect of WPC feeding on cancer cells.

Most recently (3) a group of French scientists confirmed in vitro adirect inhibitory effect of WPC on human cancer cells. Indeed similarstudies in vitro with human breast cancer cells have shown that bovineserum albumin (BSA) is the factor exerting inhibition of cancer cellreplication (4).

We have also shown that this activity of WPC is specifically dependentupon the glutamylcysteine groups (substrate for GSH synthesis) presentin the BSA fraction of WPC (U.S. Ser. No. 563,794). Interestingly, theintroduction of the cysteine delivery systemozothiazolidine-4-carboxylate (OTZ) (ozothiazolidine-4-carboxylate),while enhancing glutathione (GSH) levels in normal cells, was found toresult in feedback inhibition of the GSH cycle in human tumour cells(5). This differential effect of OTZ was recently confirmed in vivo (6).The previously described direct inhibitory effect of WPC (3) and morespecifically of BSA (4) could be explained therefore by the releaseduring incubation of a potent cysteine delivery system such asglutamylcysteine.

We have therefore reached the following conclusions:

1) BSA is the protein fraction of WPC that we found to be primarilyresponsible for the GSH promoting activity of WPC. This activity whichwe believe to be the basis for the immuno enhancing and anticarcinogeneffect of WPC, is specifically dependent upon the glutamylcysteinegroups (substrate for GSH synthesis) present in the BSA fraction of WPC.

2) The molecular weight of BSA is 66,267 hence quite different from theKW of the anti-cancer factor patented by Villadsen (MW 500-20,000). (7).

3) Our earlier findings (1,2) could be explained as follows: During DMHtreatment, WPC feeding, by increasing cellular GSH, protects the targetcells against the effects of the carcinogen. In addition, increasedavailability of substrate for GSH synthesis could inhibit replication offormed cancer cells.

4) We have now established the importance of a high level of serumalbumin (BSA) in the WPC in providing a substrate for GSH synthesis. Wecan conclude that dietary whey protein concentrate in undenatured formand containing ≧10% BSA exerts an anti-tumour effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the liver glutathione content in male mice C57BL/6NIAfed undenatured whey protein (U-Lacp), denatured whey protein (D-Lacp),casein, egg white protein or Purina diet-fed counterparts at age 10weeks, 27, 20 and 21 months.

FIG. 2 illustrates the heart glutathione content of male mice C57BL/6NIAfed undenatured whey protein (U-Lacp), denatured whey protein (D-Lacp),casein, egg white protein or Purina diet-fed counterparts at age 10weeks, 17, 20 and 21 months.

FIG. 3 illustrates the effect of various sources of whey proteinconcentrate and casein (20 g/100 g. diet) on spleen PFC response to5×10⁶ SRBC in mice.

DETAILED DESCRIPTION Definitions

(a) Whey Protein

Whey proteins are the group of milk proteins that remain soluble in"milk serum" or whey after precipitation of caseins at pH 4.6 and 20° C.The major whey proteins in cow's milk are beta-lactoglobulin (β L),alpha-lactalbumin (α L), immunoglobulin and serum albumin (SA).

The product of industrial separation of this protein mixture from wheyis called "whey protein concentrate" (WPC) or isolate. The WPC that wasused in most of our early experiments is from bovine milk (Lacprodan-80from "Danmark Protein A.S."). Use in its undenatured state is indicatedas U-Lacp, and in its denatured state is indicated as D-Lacp.Lactalbumin (L) is the term traditionally used to define WPC.

(b) c=casein;

(c) SRBC=Sheep red blood cells;

(d) PFC=Plaque forming cells (spleen): enumeration of PFC in spleen isused to assess the humeral immune response to SRBC injection;

(e) GSH=Glutathione (L-gamma-glutamyl-L-cysteinylglycine);

(f) DMH=1,2-Dimethylhydrazine;

(g) The defined formula diets tested varied only in the type of protein;

(h) Whey of bovine milk contains approximately 6 g per liter protein,most of the lactose, mineral and water soluble vitamins.

Diets Used in These Studies

Referred to below in Table 3

Diets are prepared in the following way: 20 g of selected pure protein,56 g of product 80056 protein free diet powder containing corn syrup,corn oil, tapioca starch, vitamins and minerals (Mead-Johnson Co. Inc.,U.S.A.), 18 g cornstarch, 2 g wheat bran; 0.05 g Nutramigen vit-ironpremix (Bristol-Myers, Ontario, Canada), 2.65 g KCl; 0.84 g NaCl. Thecarbohydrate and lipid components of our formula diets were the same.The only variable in the various purified diets was the type of protein(20 g protein/100 g diet). At this concentration in diet all thedifferent proteins tested provided the daily requirements of essentialamino acids for the growing mouse (8).Vitamins and minerals were thesame in each set of experiments and were added in the amount necessaryto provide daily requirements for the growing mouse (9, 10). Table 1,below, indicates the variation in suggested vitamin requirements formouse diets and their contents in some of our formulations. Thereforeall the formula diets used in our experiments were designed to provideadequate nutrition as demonstrated by normal body growth, serum protein(9) and by the absence of hair loss, dermatitis, cataract, ataxia, fattyliver etc. The latter symptoms were of course present in very old miceand were related to the aging process.

                                      TABLE 1    __________________________________________________________________________    VITAMIN AND MINERAL CONTENT OF TEST DIETS (amount/100 g diet)                           JACKSON (9)                           (range of amount                           recommended in                       TEST                           Jackson labora-                       DIETS                           torics diets)    __________________________________________________________________________    VITAMINS:                          AIN 76(10)    Vitamin A, IU               1295                   ............                       1800                           24-550      400    Vitamin D, IU               260 ............                       360 14-506      100    Vitamin E, IU               11.6                   ............                       18  1-2.7       5.0    Vitamin K, mg               0.06                   ............                       0.09                           --          0.005    Thiamine (Vitamin B1),               ....                   ............                       0.34                           0.63  0.22-.99                                       0.60    mg    Riboflavin (Vitamin B2),               ....                   ............                       0.38                           0.69  0.24-1.1                                       0.60    mg    Vitamin B6, mg               0.26                   ............                       0.36                           0.1-0.55    0.70    Vitamin B12, mg               0.0012                   ............                       0.054                           .0039-.0055 0.001    Niacin, mg ....                   ............                       5.1 9.2   26-143                                       3.0    Foilc add, mg               0.063                   ............                       0.12                           0.05-0.27   0.2    Pantothenic acid, mg               1.93                   ............                       3.38                           1-5.5       1.6    Biotin, mg ....                   ............                       0.031                           0.058 0.019-0.165                                       0.02    Vitamin C, mg               53.3                   ............                       65  --          --    Choline, mg               ....                   ............                       44  76    49-145                                       100    Inositol, mg               ....                   ............                       19.8                           19.8  --    --    MINERALS:                          AIN 76    Calcium, mg               ....                   ............                       430 #           520    Phosphorus, mg               260 ..........#         400    Magnesium, mg               63.2                   ..........#         50    Iron, mg   ....                   ............                       7.9             3.5    Zinc, mg   ....                   ............                       3.57                           #           3.0    Copper, mg ....                   ............                       0.47                           #           0.60    Iodine, mg ....                   ............                       0.023           0.02    Sodium, mg ....                   ............                       232             100    Potassium, mg               997                     360    __________________________________________________________________________     # After Minerals Analysis     (9) Hoag W. G., Dickie M. M. "Nutrition: in Green E. L. (Ed) Biology of     the Laboratory Mouse McGrawHill NY 1966 pp 39-43. (Jackson was our     supplier).     (10) The Mouse in Biomedical Research, vol III Eds FosterH. L., Seall J.     D., Fox J. B., Academic press 1983, NY pp 57-58

Immunization for Plaque Assays

The diet-fed mice were immunized by an intravenous injection of 5×10⁶washed sheep red blood cells obtained weekly from InstitutArmand-Frappier, Laval des Rapides, Quebec, Canada.

Plaque Forming Cell (PFC) Assay

The method used for assaying IgM plaque forming cells was essentiallythe one described by Cunningham and Szenberg (11), with certain minormodifications. Spleen cell suspensions were prepared by gently tampingthe spleen through a 50-mesh stainless steel screen, and collecting thecells in balanced salt solution (BSS) supplemented with 10%heat-inactivated calf serum (Grand Island Biological Company, Montreal,Quebec, Canada). The spleen cells were washed and made up to 15 ml withBSS. Sheep red blood cells were washed twice and made up to a 20%concentration. Guinea pig serum (Grand Island Biological Company,Montreal, Quebec, Canada) as a source of complement was diluted 1/15with BSS. All stock solutions were kept on ice water until used. Thetest consisted of mixing 0.05 ml of spleen cells, 0.15 ml of sheep redblood cells and 0.75 ml of the complement solution in a test tube at 37°C. The whole mixture was immediately withdrawn and put into slidechambers, sealed with warm paraffin wax, and incubated at 37° C. for 45to 60 min. The number of plaque forming cells was counted and theirtotal number per spleen estimated by multiplying the number of plaqueforming cells in each sample (0.05 ml spleen cells) by 300. The valuesare expressed per total organ rather than per 10⁶ spleen cells, sincethis appears to reflect more accurately the functional status of thespleen per se.

Mice were assayed for the plaque forming cell response to sheep redblood cells normally on the fifth day after immunization when theresponse was shown to peak or, in the kinetic study, on days 3, 4, 5 and6 post-immunization.

Statistics

The mean plaque forming cell values were compared among the dietarygroups using either Student's test, when two groups were being compared,or the analysis of variances (ANOVA) for more than two groups. Becauseof the heterogeneity of variances among groups, the adjustment given byBrown and Forsythe was used.

Spleen Glutathione Content

Ninety milligrams of mouse spleen were weighed using a Mettler PM-300balance and samples varied from 90 mg by less than 5 mg (5%). Thesamples were then homogenized in 5-sulfosalicylic acid (5% w/v).Homogenates were centrifuged for 5 min in a microfuge at 10,000 x g. Theassay was carried out using the supernatants on the same day accordingto the methods of Anderson (12). Values are expressed as μmol per g/wettissue.

Tissue Glutathione Assay

Ninety milligrams of mouse heart or liver were homogenized in5-sulfosalicylic acid (5% w/w). Homogenates are centrifuged for 5minutes in a microfuge at 10,000 x g. The assay is carried out using thesupernatants on the same day according to the method of Anderson (12),Values are expressed as μmol/g wet tissue.

After three months on either diet initiated at age 17 months, GSHcontent was found to be higher in the liver and heart of U-Lacp(undenatured whey protein Lacprodan-80) fed mice compared to the D-Lacp(denatured whey protein Lacprodan-80), casein, egg white protein orPurina diet-fed counterparts (FIGS. 1 & 2). The GSH values in heart andliver of mice fed Purina laboratory chow was similar at age 10 weeks,17, 20, 21 months. The U-Lacp diet appears to enhance the GSH content ofheart and liver above "normal" values after 3 and 4 months of continuousfeeding (FIGS. 1 & 2).

In addition, after three weeks on the U-Lacp diet, spleen GSH content isincreased during the antigen driven clonal expansion of the lymphocytesin young adult C3H/HeN mice as compared to a decline in controls fedD-Lacp, casein or egg white protein diets. In old C57BL/6NlA mice, longterm feeding of U-Lacp diet results in a moderate but sustained increasein liver and heart GSH levels (FIGS. 1 and 2). The GSH enhancingactivity of WPC is restricted to its undenatured form (U-Lacp).

                  TABLE 2    ______________________________________    AMINO ACID COMPOSITION    (g/100 g protein)                  Whey Protein                             Egg White    Amino Acid    Concentrate*                             Protein**    ______________________________________    Aspartic acid 11.3       7.9    Threonine     7.2        4.4    Serine        6.1        7.9    Glutamic acid 20.1       14.1    Proline       6.6        3.8    Glycine       2.0        3.7    Alanine       5.4        7.6    Valine        6.5        7.8    Isoleucine    6.7        6.5    Leucine       11.2       8.8    Tyrosine      2.9        4.2    Phenylalanine 3.1        6.4    Lysine        9.5        6.0    Histidine     1.9        2.2    Arginine      2.7        5.9    Methionine    2.2        3.9    Cysteine      2.4        2.4    Tryptophan    1.7        1.5    ______________________________________     *Lacprodan-80 from Danmark Protein A/S, Copenhagen,. Denmark, 1986; used     in these experiments.     **Values calculated from "Amino Acid Content of Foods", U.S.D.A., 1957.     Values from cysteine analyzed by Sigma on samples used = 2.38 g/100 g     protein and in our laboratory = 2.4 g/100 g protein.

This property is not solely due to the high cysteine content of WPCbecause another protein source with similar cysteine content (egg white)(see Table 2) does rot exhibit this biological activity. This propertyof U-Lacp does not depend specifically on its nutritional efficiency asevaluated by body weight, serum proteins, and food consumption, butappears to depend on the primary, secondary and tertiary structure ofthe protein in its native form.

Some of the previously discussed methods of increasing intracellularlevels of glutathione concentration are either toxic (13) or dangerousowing to the risks related to the initial phase of glutathione depletion(14, 15). The methods involving the use of gamma-glutamylcysteine (16),athiazolidine (17) or glutathione esters (18) (U.S. Pat. No. 4,784,685)offer an interesting possibility for short term intervention. However,their long term effectiveness in producing sustained elevation ofcellular glutathione has not been shown, nor has the possible toxicityof their long term use been disproved. Indeed, glutathione andglutathione disulfide were found to be positive in the most commonlyused short term tests for carcinogenicity and mutagenicity (13).Relevant to our invention are recent data indicating specifically that alack of the GSH precursor, cysteine, rather than a decrease inbiosynthetic enzyme activities is responsible for the deficiency of GSHnoted in aging animals (19). Similarly, the fall in cytosolic GSH in theliver of chronic ethanol fed rats does not appear to be caused by alimitation in the capacity of gamma-glutamylcysteine synthetase activity(20).

Data in FIGS. 1 and 2 show that the concentration of liver and heartglutathione in control Purina fed mice remains very constant over time.On the other hand a moderate but sustained elevation of tissue GSH wasnoted in mice fed the nutritionally equivalent whey protein (U-Lacp)diet. Only minuscule quantities of glutathione and no breakdown productsthat can be readily attributed to glutathione are excreted in urine(21). The magnitude of change in cellular glutathione concentration thatcan be achieved may be quite limited, perhaps reflecting the criticalimportance of this molecule and the attendant tight regulatory control.Glutathione itself serves as a negative feedback on the GSH syntheticenzymes, which obviously limits cellular capacity to increase GSHconcentration (22). Glutathione reductase maintains GSH in itspredominant reduced form (≧90%). This serves both to maintain thisfunctional state and also to control cellular concentration sincereduced glutathione (GSH) cannot cross the membrane, whereas theoxidized form (GSSG) can and does afflux, resulting in decreased totalglutathione. Besides these enzymes, gamma glutamyltranspeptidase (GGT)is important in GSH metabolism. GGT serves as a salvage pathway forglutamyl moieties at the cell membrane level, passing them back into thecytosol to be used in GSH synthesis. Increased activity of this enzymehas been associated with elevated GSH concentration in a number of celllines and malignant tissues (23, 24).

It is advantageous to include Vitamin B₁ (thiamine) in a diet thatresults in elevated GSH. Thiamine is involved in the transketolasereaction of the pentose phosphate shunt yielding NADPH (GSSG is reducedback to GSH by NAPPH; GSH reductase).

Vitamin B₂ (riboflavin) is also an advantageous addition. Flavinmononucleotide (FMN) and flavin adenin dinucleotide (FAD) aresynthetized sequentially from riboflavin and are involved in GSHreductase.

Some milks, especially those from New Zealand, are low in selenium.Selenium is contained in GSH peroxidase. Mammals deficient in seleniumhave markedly decreased peroxidase activity. Therefore glutathioneformation which is advantageous for its anticancer effect requires anadequate level of selenium.

If we assume a dosage level of 60 grams undenatured whey protein as adaily intake, the recommended levels of Vitamin B₁, Vitamin B₂ andselenium are as follows:

Vitamin B₁ 1.5-2.0 mg

Vitamin B₂ 1.7-2.2 mg

Selenium Methionine 40 mcg.-60 mcg.

The effects of a small increment in cellular GSH are greater thanexpected. For example, there are many reports of human and murine tumourcell lines selected in vitro for resistance to a variety ofchemotherapeutic agents. In a number of these cell lines cellular GSH isincreased consistently by 2-fold compared to the drug sensitive parentalcell line, despite the fact that the level of drug resistance is oftenmuch greater, e.g. as much as 30-fold (24, 25, 26). In these cell lines,depletion of cellular GSH by selective inhibition of synthesis restoresdrug sensitivity to the resistant cells. This is effective only if theGSH depletion is maintained throughout the drug-treatment period.

Given the fact that cellular GSH is very tightly regulated, that a2-fold increase may be maximal, and that the effect of small incrementsin GSH may be amplified by a variety of GSH-utilizing enzymes (e.g.glutathione peroxidase, glutathione-S-transferase), the reproduciblechange in GSH concentration observed in animals fed the whey-rich dietis likely to have biological importance. The chronic nature of thisaugmentation may contribute significantly to this effect.

Our findings show that in mice fed a casein diet the number and size ofDMH induced colon carcinoma were reduced by a factor of 0.3 and 0.4respectively in comparison to Purina fed controls (Table 3, below).However, in mice fed the whey protein diet with similar nutritionalefficiency the number and size of DMH-induced colon carcinoma werereduced four fold in comparison to the Purina fed controls (Table 3,below). DMH-induced colon tumours appear to be similar to those found inhumans as far as type of lesions and chemotherapeutic responsecharacteristics are concerned (27, 28).

The superiority of the anti-cancer effect of whey protein in comparisonto casein has been reported in our previous study (1). About 80% of theproteins in bovine milk are caseins and the remaining 20% are wheyproteins (29, 30). In addition, using the traditional process ofpreparing casein, the amount of whey protein co-precipitated along withthe casein varies from about 40 to 60% of the total amount of wheyprotein present in the milk (31). Therefore it is conceivable that theminor anti-cancer effect seen with casein could be due to the relatively(to caseins) small amount of whey protein coprecipitated with it. It isapparent from the above described studies that the antitumour activityof the dairy products is in the protein fraction and more specifically,as our invention demonstrates, in the whey protein component of milk.

The beneficial effect of this dietary WPC supplement is enhanced to theextent that it is a replacement for some or all of the patient's proteinintake from other sources.

SURVIVAL STUDIES The Biological Activity is Dependent on the UndenaturedConformation of WPC

(a) Survival of Old Mice During a Limited Time Period:

Our study shows that the mean survival time, over a limited observationperiod of 6-7 months ending when 55% of male C57BL/6NlA mice were dead,is increased by about 30% in mice commenced on the undenatured wheyprotein (U-Lacp) diet at the onset of senescence (age 21 months) incomparison with "controls" fed the nutritionally equivalent Purina mousechow. The survival curve of Purina fed mice was very similar to that ofcasein diet-fed mice. However, in the subsequent four months, mice onundenatured whey protein diet were switched to a denatured whey proteinconcentrate (D-Lacp) diet. During this period, the time of death of theremaining whey protein diet-fed mice became similar to that of theircasein diet or Purina-fed counterparts. Throughout the study repeatbioassays of PFC formation confirmed the correlation between hostimmunoenhancement and undenatured state of WPC in diet as indicated inFIG. 3. In the second part of the study, when the difference betweensurvival curves began to narrow, the immunoenhancing property of WPC wasabsent although its nutritional quality was preserved (D-Lacp).Throughout the entire study no significant intergroup difference wasseen in calorie intake, and body weight. Since longevity is dependentprimarily upon the genome of the individual it is unlikely that delayedmortality over a limited period of time would have influenced overalllongevity.

                                      TABLE 3    __________________________________________________________________________    Effect of dietary milk protein on animal growth and tumour    development in A/J mice treated with the carcinogen    1,2-Dimethylhydrazine.           Whey Prot.                 Casein                       Purina                             Pur/Whey                                   Pur/Cas           28 Weeks.sup.a                 28 Weeks.sup.a                       28 Week.sup.a                             20/8 Weeks.sup.b                                   20/8 Weeks.sup.b    __________________________________________________________________________    Initial           21.7 ± 0.5                 21.5 ± 0.7                       21.9 ± 0.8                             21.9 ± 0.4                                   22.0 ± 0.7    Weight.sup.c (g)    Final  21.5 ± 0.3                 21.8 ± 0.4                       19.7 ± 0.7                             21.3 ± 1.0                                   21.0 ± 0.6    Weight.sup.c (g)    Number of           8.4 ± 1.5                 24.7 ± 3.0                       35.9 ± 2.6                             15.1 ± 3.2                                   21.7 ± 4.3    Tumours.sup.c    Tumour Area.sup.c           38.8 ± 6.4                 90.9 ± 10.6                       160.0 ± 11.4                             47.9 ± 10.4                                   77.7 ± 10.9    __________________________________________________________________________     .sup.a) Mice treated with DMH for 24 weeks, and then sacrificed 4 weeks     later.     .sup.b) Mice treated with DMH for 24 weeks, and then sacrificed 4 weeks     later. They were maintained on Purina Mouse Chow for 20 weeks and then     switched to either Whey Protein or Casein diet for the remaining 8 weeks.     .sup.c) Mean ± SEM.     ANOVA: solid line(s) connect those means not significantly different (p <     0.05).

    Group Whey              Pur/Whey                    Pur/Casein                          Casein    Number of    Tumours    Tumour    Area

However, at least in terms of the immunoenhancing effect of the diet,this study could be regarded as a single direction cross-over from test(ULacp) to control (D-Lacp) diets, showing that the biological activityof WPC on survival of old mice is dependent upon its undenatured stateand correlating directly with the PFC assay used in our study (asillustrated in FIG. 3).

(b) Short and Long Term Survival of Mice with DMH-Induced Colon Cancer:

In DMH treated mice we noticed a difference between mortality by the 28weeks end point and the survival time to the end of the experiment inrelation to dietary protein type. During the first seven months ofstudy, the mice fed undenatured whey protein (U-Lacp) had no death ascompared to a 33% mortality observed towards the end of this period inthe casein and Purina groups. In the subsequent four months mice on wheyprotein were fed denatured whey protein (D-Lacp). During this latterperiod the D-Lacp diet appeared to have no favourable effect on survivalin comparison to the casein diet (Table 4, below).

Throughout the study repeat bioassays of spleen PFC were done todocument the physiologic effects of the diets on immune function asreported previously and the stability of these effects. Theimmunoenhancing effect of the U-Lacp diet was consistently confirmed forthe first 7 months of the study; however, in the following fourmonths-(D-Lacp), the immunoenhancing effect previously observed in micefed the U-Lacp diet was absent. The values of PFC response in relationto either the U-Lacp diet or the D-Lacp diet were consistent with thosepresented in FIG. 3. This study therefore confirms the hypothesis thatthe biological activity of WPC on survival of tumour bearing mice isdependent upon its undenatured state correlating directly with the PFCassay used in our study.

                  TABLE 4    ______________________________________    Effect of dietary milk protein on short and long term    survival in A/J mice treated with the carcinogen 1,2-    Dimethylhydrazine for 24 weeks.                 DIETARY GROUP.sup.b                 Whey Protein.sup.d                            Casein  Purina    ______________________________________    Mortality.sup.a at 28 weeks                   0%             33%   33%    Survival time.sup.c in weeks                   40           41      30    ______________________________________     .sup.a significant by Chi Square analysis: Whey Protein vs. Purina vs.     Casein p<0.05.     .sup.b Originally 12 mice per group.     .sup.c Survival time in weeks from the first dose of carcinogen. Whey     protein and Casein differ significantly from Purina, MantelCox test     p<0.01.     .sup.d Undenatured Whey Protein used from weeks 3 to 28. Denatured Whey     Protein used from weeks 28 until end.

The experiment referred to above which are summarized in Tables 3 and 4used Lacprodan 80 as a source of undenatured WPC:

We now prefer to use a whey protein concentrate (WPC) in undenaturedform prepared from milk treated in the most lenient way compatible withaccepted standards of safety with regard to bacterial contamination. Theextremely high solubility index indicated that the proteins present areessentially undenatured, hence demonstrating the leniency of theultrafiltration process 31!. Although the proteins contained in theconcentrates from the other commercially available sources examined weremostly in undenatured form, as indicated by the relatively highsolubility of the concentrates, the content of serum albumin andimmunoglobulins in these mixtures is below the level activity 31!. Thesevery thermolabile proteins are denatured, hence precipitated andpartially lost from whey when high pasteurization temperatures areutilized.

Our studies also showed that administration of S(n-butyl) homocysteinesulfoximine, which reduces splenic glutathione in half, significantlyreduced the humoral immune response of whey protein-fed mice. This wastaken as further evidence for the important role of glutathione in theimmunoenhancing effect of dietary whey protein (32).

Tissue glutathione concentration may be increased by administration ofgamma-glutamyl-cysteine. Intracellular glutathione increased in thekidney by about 50%, 40-60 minutes after subcutaneous (s.c.) injectionin mice, returning to control values 2 hours later (33). Theadministered gamma-glutamylcysteine is transported intact into thecells, and serves as a substrate for glutathione synthetase (33).

Advances in amino acid sequencing of food proteins allowed us toinvestigate the occurrence of glutamylcysteine groups in whey proteinand the possible relation to glutathione promotion. Indeed, whey proteinconcentrate from bovine milk contains substantial amounts ofglutamylcysteine groups, unlike casein, which does not increase tissueglutathione when fed to mice (35). The glutamylcysteine groups arelocated primarily in the serum albumin fraction (six groups/molecule).Glutamylcysteine groups are extremely rare in animal and plant edibleproteins. Extensive search of all available data on amino acidsequencing of edible proteins reveals that the Glu-Cys group with adisulfide link is indeed limited to some of the whey protein, and to theovomucoid fraction of egg white which contains 2 of these groups in a30,000 mol.wt.molecule (31).

Our recent (31) data further indicate that the humoral immune responseis highest in mice fed a dietary whey protein concentrate exhibiting thehighest solubility (undenatured conformation) and, more importantly, agreater relative concentration of the thermolabile bovine serum albumin(≧10%) and immunoglobulins. In addition, the mice fed this type of wheyprotein concentrate exhibit higher levels of tissue GSH. Theglutamylcysteine groups (rare in food protein) and the specificintramolecular bond as related to the undenatured conformation of themolecule are considered to be key factors in the glutathione-promotingactivity of the protein mixture.

Recent experiments in Japan 36! showed that spleen cells of BALB/c malemice fed 25 g of our undenatured whey protein concentrate (WPC) (forwhich the Trademark name "Immunocal" has been applied) per 100 g dietfor 4 weeks had an increased immune response to SRBC in vitro and a highcontent of L3T4+ cells (12.58×10⁶ ±1.36) than mice fed an isocaloricdiet with 25 g. pure casein/100 g. diet (3.69×10⁶ ±0.50). Similarly, thespeen L3T4+/LYt-2+ ratio was 1.36±0.07 in undenatured WPC fed mice and0.55±0.07 in casein-fed controls (P<0.001). Conversely, the relativelyhigh concentrations of the thermosensitive serum albumin andimmunoglobulins resulting from the low degree of pasteurization of milkin our WPC, may reflect more closely the pattern of raw milk. These datalend support to the hypothesis that the thermolabile Glu-Cys containingproteins such as serum albumin in undenatured conformation are crucialelements for the biological activity of whey protein concentrate.

Suitable bovine whey protein concentrate (WPC) has been prepared by the"Service de recherche sur les aliments du Ministere de l'agriculture duQuebec" in St-Hyacinthe, Quebec, Canada, with the followingcharacteristics: pure protein content 75% (the rest mostly lactose, somefat and moisture); solubility index: (pH 4.6); 99.5%. Proteincomposition as of total whey protein measured by polyacrylamide gelelectrophoresis (31) was: beta-lactoglobulin 59.1±4.0;alpha-lactalbumin: 6.6±0.7; serum albumin: 9.7±1.0; immunoglobulin24.6±2.6 (mean±SD). The solubility index should preferably be above 99%.

The serum albumin of about 10% of the total whey protein was almosttwice the corresponding value found in other commercially available wheyprotein concentrates that have been examined. It is believed that aserum albumin level ≧10% is highly advantageous to improving the immunesystem.

Serum albumin includes a substantial amount of glutamyl cysteine whichis a substrate for glutathione synthesis in the body. The role ofglutathione is discussed in detail in or article "The BiologicalActivity of Undenatured Dietary Whey Proteins: Role of Glutathione",Clin. Invest Med 14: 296-309, 1991 (31), which is incorporated byreference in its entirety.

Immunoglobulin in the range of about 25 to 30% of total whey protein isalso important. Pasteurization at 72° C. for 13 seconds resulted in animmunoglobulin level of 28±2%. We have found it possible to achieve aserum albumin level as high as 14±1% with milk pasteurized at 72° C. for13 seconds.

Upon bacteriological analysis no staph, salmonella, B cereus, or E coliwere isolated in either the WPC prepared by the "Service de recherchesur les aliments du Ministere de l'agriculture du Quebec" or in thesample pasteurized at 72° C. for 13 seconds. Other samples were preparedby heating the milk to 63° C. for 30 minutes, with good results.

30 ml of heparinized blood may be used to determine the glutathionecontent of blood mononucleated phosphate buffered saline adjusted sothat there are 10⁷ cells per tube. After centrifugation 900 ml of wateris added to the pellet to lyse all the cells. To each aliquot is added30% sulfosalicylic acid for a final concentration of 3% in 1 ml. After15 minutes incubation, the samples are centrifuged, and the clearsupernatant is used for the biochemical assay according to the method ofAnderson 37!. Values are expressed as nanomol (nMol) per GSH/10⁷ cells.Blood lymphocyte subsets may be determined by flow-cytometry.

The total serum protein, including the albumins and the immunoglobulinsmay be determined by the Biuret method. The level of Immunoglobulin A(IgA), Immunoglobulin G (IgG) and Immunoglobulin M (lgM) may be measuredby immunonephlometry.

The presence of glutamylcysteine groups in the serum albumin componentof the whey protein concentrate is considered to be a key factor in theglutathione-promoting and immunoenhancing activity of the proteinmixture of the undenatured WPC. Our laboratory studies indicate thatwhey protein concentrates from other sources did not produce significantbiological activities even though they exhibited similar nutritionalefficiency. The percent serum albumin concentration in these products is(as mean±SD) respectively: 4±1 in Promod (Ross laboratories), 4±1 inAlacen 855 (New Zealand Dairy), 4.8±1 in Lacprodan-80 (produced from1989 by Danmark Protein), 4±0.1 in Sapro (Sapro, Montreal), 4±1 inSavorpro-75 (Golden Cheese, Calif., USA), 5±1 in Bioisolate (Lesueur,Isolates, Minn.) 8! and 4.3±1 in Promix (Dumex, Quebec). Similarly, thecontent of the other thermolabile protein, immunoglobulin, was abouthalf the value of the undenatured WPC used in this study.

The results indicate that undenatured whey proteins by providingspecific fuel for glutathione replenishment in the immunocytes couldrepresent an adjuvant to other forms of therapy.

Historically, and up until now, bacteria and spores in milk were reducedby thermal treatment (pasteurization). In order to be effective, thatmethod inevitably produced denaturation, and hence subsequentprecipitation and loss in the curd of a substantial amount of the mostthermolabile and presumed biologically active fractions of serum albuminand immunoglobulin.

Our objective is to obtain a whey protein concentrate (w.p.c) containingthe proteins in proportion and conformation as close as possible to thatof raw milk, compatible with accepted safety standards of bacterialcontent. Up until now we have utilized the lowest acceptable level ofheat treatment of milk in order to preserve thermolabile whey protein.

An alternative to heat treatment is a method based on membranemicrofiltration. Utilizing Bactocatch (Alfa-Laval Ltd. Scarborough,Ontario) we can obtain a permeate by special membrane microfiltration ofthe skimmed milk whose bacteria content has been reduced to less than0.5% of original input levels.

This permeate is then treated with rennet and the proteins in the wheysupernatant concentrated by a lenient procedure to obtain the desiredundenatured whey protein concentrate. The membrane microfiltrationconcept is an alternative to the heat treatment of milk as anappropriate way to preserve heat labile whey proteins, althoughtechniques and equipment may be improved in time.

Tables 5 and 6 illustrate schematically a process to produce an improvedundenatured WPC which we have referred to under the trademark Immunocal.Table 7 is a comparative chart showing the characteristics of Immunocalin comparison with the sources of WPC and showing also the consequencesof 3 weeks dietary treatment.

We have concluded as a result of our work that undenatured whey proteinconcentrate (WPC) is of value in the prophylaxis of cancers, such aschemically induced cancer, typically such as colon cancer which ispromoted by carcinogens such as dimethylhydrazine. It also is useful forthe treatment of patients having cancer cells such as chemically inducedcancer cells, to inhibit the replication of such cells. An approximatedosage for humans is in the range of about 8 to 40 grams daily andpreferably 20 to 40 grams daily. It is particulary beneficial toadminister 30 to 40 grams daily. It has been established that it isparticularly advantageous to use WPC having a serum albumin inconcentration of at least 10%±1. The serum albumin should be at least 9%and most preferably at least 9.5%.

As the published literature shows, tumors of the colon induced bydimethylhydrazine in experiments with mice are similar to cancer of thecolon in humans in terms of the type of lesion and response tochemotherapy. (27,28).

The discovery that the granulocytes/lymphocytes (G/L) ratio is increasedin terminal cancer cases and correlates with the patient's condition(38) has prompted an interest in the factors that might influence thisimportant parameter.

                  TABLE 5    ______________________________________    A SCHEMATIC REPRESENTATION OF THE PROCESS TO    PRODUCE THE WPC WHICH WE PREFER TO AS IMMUNOCAL    ______________________________________    Raw milk       ↓    Skimmed at 35° C.                     = → cream       ↓           Skimmed milk pasteurized at 63° C. for 30 minutes.              ↓    At 38° C.:           Addition of rennet (20 ml/100 kilos), allowing the           agitation to resolve at low speed.       ↓      = → curd    Whey       ↓    Filtered with cheese cotton to remove debris (45 minutes):    At 40° C.:           Ultrafiltration (Romecon UFSI, polysulphone membrane,           cut off 50,000, pore diameter 0.06 of an inch, surface           2-3 m.sup.2).    :      Diafiltration to wash out salts and lactose.           Whey Protein Concentrate              ↓    Pasteurized at 63° C. for 30 minutes.              ↓    At 40° C.:            Lyophylization (16 hours).              ↓           Whey Protein Concentrate, Powder:    ______________________________________

                  TABLE 6    ______________________________________    ASSAY OF PRODUCT OF TABLE 5    ______________________________________    Polyacrylamide                  Serum Albumin                              β-Lactoglobulin    gel electrophoresis                  10 ± 1%  57.8 ± 0.9%    α-Lactalbumin                  Immunoglubin    11.4 ± 0.6%                  22 ± 0.7%    ______________________________________

For example granulocyte depletion using extracorporeal circulation wasfound to significantly reduce the size of a transplanted tumor in rabbit(39). Recent experimental studies in mice suggested the possibility thatthe second parameter of the G/L equation could be influenced by dietarymeans. Mice fed whey protein concentrate prepared so as to preserve themost thermosensitive molecule of whey, such as serum albumins in theirnative undenatured form, exhibited an enhanced immune response (35)manifested, in part, by an increased resistance to pneumococcalinfection (40,1) and carcinogen induction of cancer (1)

This is the product which we referred to as undenatured WPC. Increasedimmune reactivity of mice fed with undenatured WPC was found to persistin naive cells transferred in vitro (36). The serum albumin contains sixglutamylcysteine groups (31) hence provides the specific substrate forthe synthesis of glutathione (33, 34) (GSH). These groups are extremelyrare in food proteins (31).

                                      TABLE 7    __________________________________________________________________________    PHYSICAL-CHEMICAL CHARACTERISITCS AND BIOLOGICAL ACTIVITY OF DIFFERENT    TYPES OF WHEY PROTEIN CONCENTRATES           Undenatured           conformation                  Protein composition (% of                                      Effect of 3 weeks dietary treatment           Solubility                  total whey proteins)                                 Immuno-                                      PFC.sup.4                                            Glutathione (μmol/g).sup.5           index (pH 4.6)                  β-LG.sup.a                       α-La.sup.2                            SA.sup.3                                 globulins                                      × 10.sup.-3                                            Liver Heart    __________________________________________________________________________    Immunocal           99.5%  57.8 ± 0.9                       11.4 ± 0.6                            10 ± 1                                   22 ± 0.7                                      148 ± 16                                            7.95 ± 0.40                                                  1.15 ± 0.7    Promod 97%    61 ± 3                       20 ± 1                            4 ± 1                                 15 ± 3                                      65 ± 14                                            6.64 ± 0.41                                                  1.0 ± 0.7    Alacen 855           97.1%  62 ± 8                       22 ± 3                            4 ± 1                                 12 ± 6                                      66 ± 17                                            6.04 ± 0.36                                                  --    Lacprodan-80           96%    62 ± 4                       18 ± 2                            4.8 ± 2                                 15 ± 4                                      44 ± 15                                            6.70 ± 0.20                                                  --    Sapro  95%    60 ± 3                       21 ± 2                            4 ± 1                                 14 ± 3                                      31 ± 8                                            --    --    Savorpro-75           98%    63 ± 3                       20 ± 1                            4 ± 1                                 13 ± 3                                      31 ± 8                                            --    --    Biolosolate           90.1%  66 ± 4                       15 ± 1                            5 ± 1                                 12 ± 3                                      65 ± 20                                            --    --    Casein --     --   --   --   --   35 ± 9                                            --    1.0 ± 0.8    __________________________________________________________________________     Values are expressed as mean ± S.D.; 20 g protein/100 g diet.     .sup.1 LG, Lactoglobulin     .sup.2 La, AlphaLactalbumin     .sup.3 S.A., Serum Albumin2     .sup.4 Number of plaqueforming cells/spleen 5 days following immunization     with a 5 × 10.sup.6 sheep red blood cells     -- Signifies not done     *Please see test for statistical reference.

The favourable effect of undenatured WPC in animal experiments wereassociated with a sustained elevation of tissue glutathione levels (41).However, nowhere this effect was more evident than in the spleen cellsduring the antigen driven clonal expansion of the lymphocyte pool (32):these experiments clearly indicate that the immune enhancing activityi.e. increased lymphocyte proliferation of dietary undenatured WPC isrelated to the observed greater production of GSH in the lymphocytes incomparison to an actual GSH decrease noted in casein fed controls duringa similar antigenic challenge (32). GSH, a tripeptide thiol found invirtually all cells, is the major free radical oxygen scavenger (42).More specifically, the GSH content of the lymphocytes appears to becorrelated with the ability of oxidants and thiols to modulate theirproliferation (43). In this sense modulation of intracellular GSH mayaffect immune responsiveness (44, 45).

These experiments further indicate that the enhancing effect of dietarywhey proteins on the lymphocytes is particularly evident when theantigenic stimulus for active proliferation is present.

However, this unique relation between dietary undenatured WPC andlymphocytes production is also manifest in normal unimmunized mice.After three weeks of dietary regimen, the spleen weights, as well as thespleen weight: body weight ratio of mice fed 20 g or 30 g undenaturedWPC/100 g diet were moderately higher than the corresponding values ofmice fed nutritionally equivalent diets containing 20 g or 30 g ofeither casein, soy protein or fish protein/100 g diet (46). Morespecifically, the number of cells per spleen were higher in undenaturedWPC fed mice than the corresponding values in mice fed equivalentamounts of casein, wheat, soy and fish protein (35).

A more detailed study revealed that the number of nucleated cells in thespleen of mice fed a 25% undenatured WPC diet was significantly higherthan the corresponding values in mice fed a control 25% casein diet.Interestingly, the observed changes were specifically related todoubling of the T cell population: particularly the L3T4⁺ subpopulationincreased four fold, whereas the number of B-cells was unchanged (36).This later finding is consistent with other studies showing that theeffects of undenatured WPC are not exerted centrally on the rate ofprimary B-lymphoctye production in the bone marrow and that the immuneenhancing effect is specifically seen in response to T-cell dependentantigens (47).

The above described effect of undenatured WPC on the lymphocytes maywell explain the observed inhibitory effect of this form of dietaryregimen on experimental tumors. Recent experiments showed thatcontinuous feeding of undenatured WPC in the diet inhibits thedevelopment (number and size) of tumors in the colon of mice over aperiod of 24 weeks of dimethylhydrazine (DMH) treatment (1). Thisantitumor effect could be caused by increased resistance of target cellsto the carcinogen and/or a direct inhibitory effect of dietaryundenatured WPC on tumor growth. A subsequent series of experiments (2)where animals were fed standard laboratory diet for the first 20 weeksof DMH and then switched to the undenatured WPC diet for the remaining 8weeks of DMH treatment, clearly indicate an inhibitory effect ofundenatured WPC feeding on cancer cells.

It is noteworthy that the enhancing effect of undenatured WPC on theperipheral lymphoid tissues of normal animals is not associated withsignificant changes in the total number of blood lymphocytes (36,46).The absence of variation in total number of circulating lymphocytes wasalso noted in healthy humans (Otsuka Pharmaceutical Co. Ltd.).

The induction of cellular GSH synthesis by the glutamylcysteine groupsof undenatured WPC may constitute another interesting effect of thisproduct in cancer patients. Indeed, the introduction of the cysteinedelivery system OTZ (ozothiazolidine -4-carboxylate), while enhancingGSH levels in normal cells was found to result in feedback inhibition ofthe GSH cycle in human tumor cells (5).

A clinical trial was arranged in Japan by Otsuka Pharmaceutical Co. Ltd.The clinical team was directed by Dr. Masakazu Adachi. The clinicaltrial involved five patients who had an advanced stage terminal cancer.A summary report is reproduced below as Table 8 and laboratory data ofhematocytes and glutathione levels are given in Table 9 for four of thepatients. The dosage level for each patient was 30 grams per day.

As shown in Table 8 the lymphocyte number in all patients increasedduring the adminstration of the undenatured whey protein concentrate.Two other patients with an advanced stage had withdrawn from the trialbecause of digestive problems. Previous investigation had reported thatonce the lymphocyte number had dropped in cancer patients, the recoveryof the lymphocyte number to a normal level may not be possible. Theincrease of the lymphocyte number is of importance because of itspotentiality for improving the G/L ratio.

The results shown in Tables 8 and 9 may be contrasted with healthyindividuals who (based on tests of 14 individuals) will usually have anglutathione PMN level of 0.34±0.02, a glutathione RBC level of0.066±0.006, a WBC of 6640±1700, a lymphocyte number of 2200±460 a G/Lratio of 1.85±0.45 and Plt 25.4±3.3.

Table 10 shows the bacteriological analysis and also the proteindistribution and solubility of the undenatured WPC used in the trialsdescribed in Tables 8 and 9.

In conclusion, it appears that the administration of WPC to cancerpatients increases the number of blood lymphocytes and decreases the G/Lratio. The lymphocyte concentration, very low at the outset, tends toreturn towards normal values. On the basis of previously describedexperimental evidence (38,39), the observed effect of WPC suggestsinhibition of cancer cell growth.

In tests of undenatured WPC subsequent to those described in Tables 8and 9, the following observations were made:

(1) Patient F had solid tumors in her abdomen that could be identifiedby physical examination and she had received chemotherapy. She haddifficulty in consuming the undenatured WPC and was about to withdrawfrom treatment. However it was found that as long as she was taking theundenatured WPC her lymphocyte number increased, and the increasingnumber of tumor markers even showed signs of decreasing.

                                      TABLE 8    __________________________________________________________________________                       improvement or terminal stage                                       during undenatured WPC                                       administration:    Patient        Cancer              Recurrence                       exacerbation    G/L   lymphocytes                                                   GSH/lymphocytes                                                             period    __________________________________________________________________________    A   Gastric              peritonitis                       tumor ↑                                (+)    n.c.  980 → 1400                                                   n.t.      54 days              carcinomatosa                       ascites ↑                                ileus                        drop out    B   Uterus              primary  scc. 26 → 100                                liver  n.c.  960 → 1300                                                   0.36 → 0.47                                                   → 0.34                                                             143 days        lung meta.              tumor    p.s.: 0  metastasis ↑           unfavourable    C   Breast              bone meta.                       improveoment    4.4 → 2.2                                             480 → 1250                                                   0.33 → 0.43                                                             133 days        bone meta              (radiation therapy)                       p.s.: 0                               favourable    D   Lung  LN meta. tumor ↑                                (+)    11.6 → 4.0                                             340 → 930                                                   0.85 → 0.53                                                             79 days    brain meta              bone meta                       disturbance of consiousness   died 50 days later after                                                     drop out    E   Esophag.              --       lung metastasis                                (+)    n.c.  600 → 1000                                                   0.26 → 0.46                                                   → 0.27                                                             56 days        LN meta.                                             unfavourable    __________________________________________________________________________

                                      TABLE 9    __________________________________________________________________________    Lab Data of Hematocytes and Glutathione Level: Cancer    Pre Immu         After              Patient                  Cancer                       Compli.                           GSH/PMN                                 GSH/RBC                                      WBC                                         Lymp.                                             Gran.                                                G/L                                                   RBC                                                      Plt.                                                         Mono.    __________________________________________________________________________    0 day     C   Breast                       L.C.                           0.33  0.075                                      26.9                                         4.8 20.9                                                4.4                                                   356                                                      9.5                                                         1.2         8 days              C   Breast   0.47       25.1         35 days              C   Breast   0.40  0.083                                      34.3                                         8.3 23.4                                                2.8                                                   352                                                      11.5                                                         2.6         77 days              C   Breast   0.43  0.095                                      32.6                                         8.1 22.l                                                2.7                                                   357                                                      10.0                                                         2.4         91 days              C   Breast   0.37  0.074                                      40.4                                         11.6                                             25.7                                                2.2                                                   362                                                      12.0                                                         3.1         119 days              C   Breast         0.088                                      38.6                                         16.1                                             19.2                                                1.2                                                   367                                                      11.4                                                         3.3         133 days              C   Breast   0.31  0.090                                      40.0                                         12.5                                             24.5                                                2.0                                                   359                                                      10.7                                                         3.0    26 days   E   Esophag. 0.26  0.067                                      38.5                                         5.9 29.4                                                5.0                                                   416                                                      23.6                                                         3.2         0 days              E   Esophag. 0.46  0.078                                      48.2                                         6.8 38.6                                                5.7                                                   401                                                      24.8                                                         2.8         14 days              E   Esophag. 0.39  0.061                                      45.5                                         7.4 34.6                                                4.7                                                   405                                                      26.0                                                         3.5         42 days              E   Esophag.       0.066                                      53.6                                         8.7 40.6                                                4.7                                                   393                                                      25.1                                                         4.3         56 days              E   Esophag. 0.27  0.066                                      57.8                                         0.5 43.0                                                4.1                                                   408                                                      25.1                                                         4.3    5 days    D   Lung     0.85  0.073                                      44.7                                         3.4 39.3                                                11.6                                                   345                                                      32.9                                                         2.0         15 days              D   Lung     0.46  0.072                                      51.3                                         6.2 39.5                                                6.4                                                   292                                                      28.2                                                         5.6         42 days              D   Lung     0.53  0.071                                      50.6                                         9.3 37.3                                                4.0                                                   325                                                      32.9                                                         4.0    3 days    B   Uterus   0.38  0.083                                      45.8                                         9.6 33.6                                                3.5                                                   319                                                      19.1                                                         2.6         10 days              B   Uterus   0.34  0.083                                      42.8                                         7.7 32.9                                                4.3                                                   306                                                      17.9                                                         2.2         18 day              B   Uterus   0.50  0.087                                      57.5                                         12.1                                             42.4                                                3.5                                                   575                                                      21.7                                                         3.0         45 days              B   Uterus   0.55  0.090                                      51.0                                         13.1                                             35.5                                                2.7                                                   315                                                      19.8                                                         2.4         87 days              B   Uterus   0.43  0.099                                      52.2                                         10.1                                             37.1                                                3.7                                                   324                                                      19.2                                                         5.0         101 days              B   Uterus   0.47  0.076                                      66.5                                         12.9                                             49.5                                                3.8                                                   296                                                      28.8                                                         4.1         129 days              B   Uterus         0.086                                      72.0                                         13.7                                             53.0                                                3.9                                                   258                                                      28.6                                                         5.3         143 days              B   Uterus   0.34  0.092                                      77.3                                         13.3                                             58.7                                                4.4                                                   267                                                      30.9                                                         5.3    __________________________________________________________________________

                                      TABLE 10    __________________________________________________________________________    Undenatured WPC powder preserved at 4° C. in dry environment with    3 exposures at room    temperature (22-25° C.) for about 4 days.    __________________________________________________________________________    BACTERIOLOGICAL ANALYSIS    Time after production                Staph. Salmonella                             B cereus                                   E coli                                       Coliforms    __________________________________________________________________________    4 days      0      0     0     0   <100/100 g    14 months   0      0     0     0   <100/100 g    __________________________________________________________________________    Protein distribution (1) and solubility (2)    Time after production              Solubility                   Beta-Lactoglubulla                            Alpha Lactalbumin                                     Serum albumin                                            Others (3)    __________________________________________________________________________    4 days    98.9 59.1 ± 4.0 (4)                            6.6 ± 0.7                                     9.7 ± 1.0                                            24.6 ± 2.6    14 months 99.0 58.9 ± 3.9                            9.6 ± 1.1                                     9.6 ± 1.1                                            22 ± 2.9    __________________________________________________________________________     (1) Months by polyacrylamide gel electrophoresis     (2) Solubility index (ph 4.6)     (3) Mostly immunoglobulin     (4) Mean ± SD

(2) Patient G was treated for a local recurrence of cancer afterradiation. She received radiation treatment in November, 1992.Administration of undenatured WPC commenced December 4. Her lymphocytesimproved between December 3 and Jan. 20, 1993 from 960 to 1310. As ofFebruary 3 there was no recurrence and her lymphocytes were 1370 and G/L3.1.

(3) Patient H had received radiation therapy for a local tumor.Administration of undenatured WPC was commenced Dec. 16, 1992. At thattime, test results were as follows:

    ______________________________________            WBC  2700            Lym.  480            G/L  4.6            P/t  9.5    ______________________________________

Results on December 24 were:

    ______________________________________            CEA  11.0            WBC  4320            Lym.  910            G/L   3.7            P/t  10.7    ______________________________________

On January 20 results were:

    ______________________________________            CEA   7.1            WBC  3420            Lym.  830            P/t  11.5    ______________________________________

On February 3 the results were:

    ______________________________________            CEA  5.2            WBC  3,300            Lym.   860            G/L  2.5            P/t  11.5    ______________________________________

It was noted that progress of the patient was good.

(4) Patient "I" had received radiation therapy on mediastinum, oncervical and total brain. Administration of undenatured WPC was startedon Dec. 1, 1992. His lymphocytes increased from 340 on November 26 to930 on January 13. His G/L ratio decreased from 12.1 to 4.4 and WBCincreased from 4500 to 5100.

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The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of prophylaxisof colon cancer of the type induced by dimethylhydrazine in mammalscomprising the administration to a patient of an undenatured wheyprotein concentrate containing at least 9.5% serum albumin, at a dailydosage in the range 8 to 40 grams.
 2. A method for treating cancer,comprising administering to a patient having cancer an undenatured wheyprotein concentrate containing at least 9.5% serum albumin in amountsufficient to increase the lymphocyte number of the patient.
 3. A methodfor treating cancer, comprising administering to a patient having canceran undenatured whey protein concentrate containing at least 9.5% serumalbumin in an amount sufficient to decrease the G/L ratio of thepatient.
 4. The method of any one of claims 2 or 3 in which the dailydosage level is in the range of 8 to 40 grams.
 5. The method of any oneof claims 2 or 3 in which the undenatured whey protein concentrate isprepared from milk and contains substantially all the proteins presentin the raw milk.
 6. The method of any one of claims 2 or 3 includingadministering, in addition, Vitamin B₁ in the amount of about 1.5 to 2.0mg per 60 grams of undenatured whey protein concentrate.
 7. The methodof any one of claims 2 or 3 including administering, in addition,Vitamin B₂ in the amount of about 1.7 to 2.2 mg per 60 grams ofundenatured why protein concentrate.
 8. A method of treating cancer in apatient in need thereof comprising the administration to said patient ofa substantially undenatured whey protein concentrate, which concentratecomprises substantially all thermolabile whey proteins of the sourcemilk with glutamylcysteine groups and disulfide bonds, in an amountsufficient to replenish depleted intracellular glutathione, enhanceimmune response and exert anti-cancer effects.
 9. A method of treatingcancer in a patient in need thereof comprising the administration tosaid patient of a substantially undenatured whey protein concentrate,which concentrate comprises substantially all thermolabile whey proteinsof the source milk with glutamylcysteine groups and disulfide bonds, inan amount sufficient to increase the lymphocyte number.
 10. A method oftreating cancer in a patient in need thereof comprising theadministration to said patient of a substantially undenatured wheyprotein concentrate, which concentrate comprises substantially allthermolabile whey proteins of the source milk with glutamylcysteinegroups and disulfide bonds, in an amount sufficient to decrease the G/Lratio of a patient having cancer.
 11. A method of treating cancer in apatient in need thereof comprising the administration to said patient ofa substantially undenatured whey protein concentrate, which concentratecomprises at least about 2.4 grams cysteine per 100 grams protein, in anamount sufficient to replenish depleted intracellular glutathione,enhance immune response and exert anti-cancer effects.
 12. A method ofclaim 8, in which the serum albumin is at least 9.0%.
 13. A method as inany one of claims 8 to 12, in which the daily dosage level is in therange of 8 to 40 grams.
 14. A method as in claim 13, in which the dailydosage level is in the range of 20 to 40 grams.
 15. A method as in claim14, in which the daily dosage level is in the range of 30 to 40 grams.16. A method of treating cancer in patients having lesions of the colonof the type induced by dimethylhydrazine and being in need of treatmentcomprising the administration to said patient of a substantiallyundenatured whey protein concentrate, which concentrate comprisessubstantially all thermolabile whey proteins of the source milk withglutamylcysteine groups and disulfide bonds, in an amount sufficient toreplenish depleted intracellular glutathione, enhance immune responseand exert anti-cancer effects.
 17. A method as in any of claims 8 to 11or 16 above, in which selenium is administered in the amount of about40-60 mcg (calculated as selenium methionine) per 60 grams ofundenatured whey protein concentrate.
 18. A method as in any one ofclaims 8 to 11 or 16 above, in which Vitamin B₁ is administered in theamount of about 1.5-2.0 mg per 60 grams of undenatured whey proteinconcentrate.
 19. A method as in any one of claims 8 to 11 or 16 above,in which Vitamin B2 is administered in the amount of about 1.7-2.2 mgper 60 grams of undenatured whey protein concentrate.
 20. A method as inany one of claims 2, 3, 8 to 11 or 16 above, in which the following areadministered per 60 grams of undenatured whey proteinconcentrate:Vitamin B₁ about 1.5-2.0 mg Vitamin B₂ about 1.7-2.2 mg andSelenium 40-60 mcg (calculated as selenium methionine).
 21. A method ofprophylaxis of colon cancer of the type induced by dimethylhydrazine inmammals comprising the administration to a patient of an undenaturedwhey protein concentrate, which concentrate comprises substantially allthermolabile whey proteins of the source milk with glutamylcysteinegroups and disulfide bonds, at a daily dosage in the range of 8 to 40grams.
 22. A method as in claim 20 in which the daily dosage level is inthe range of 8 to 40 grams.